Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) is a technique used for the quantification and detection of bio-molecules and other macro-molecular substances for applications ranging from proteomics and cancer early detection to forensic investigations. Especially for proteomics and cancer research, sensitivity and analyte concentration are essential for successful measurements, since the analyte is often only available in very small quantities and/or high dilution.
The described invention aims at increasing the reproducibility and sensitivity of MALDI-MS for water-insoluble matrix based samples through improving the sample preparation process. MALDI-MS is an advanced mass spectrometry technique used to detect large molecules (“macro-molecules”). Such molecules cannot be measured using conventional mass spectrometry techniques due to fragmentation. MALDI-MS achieves ionization by proton transfer from a matrix compound (usually a crystal-forming acid) to the analyte to be analyzed. To achieve this proton transfer, the analyte needs to be embedded within the matrix compound, which generally exceeds the analyte amount by two to three magnitudes. This is achieved by creating a mixed solution of both analyte and matrix, which is drop-deposited on a sample plate. Evaporation results in a solid residue of analyte/matrix compound. This solid residue is then ablated with a laser focused into a tight (˜100 μm diameter) high-intensity spot. The ablated material forms a gaseous cloud above the sample in which protons are transferred from matrix to analyte, resulting in charging of the analyte molecules, which can subsequently be analyzed in the mass spectrometer by use of electrical or magnetic fields.
The standard matrix materials used in MALDI investigations can be generally divided into water-soluble and water-insoluble compounds. The mostly used water-soluble compounds are 2,5-dihydroxybenzoic acid (2,5-DBH) and 3-hydroxypicolinic acid (3-HPA), while the most popular water-insoluble material is α-cyano-4-hydroxycinnamic acid (HCCA).
Since drop-deposition of matrix/analyte solution on a flat plate typically yields irregular circular deposits, such deposits are difficult to analyze. Usually a trial and error procedure is used to find a “sweet spot” that yields a good signal-to-noise ratio. This is time consuming, and yields poorly reproducible data. Consequently, this has led to the invention of so-called anchor plates, where arrays of small (100-800 μm diameter) hydrophilic spots are created on a hydrophobic substrate. This allows deposited droplets to anchor to the hydrophilic spots, since they are repelled by the hydrophobic surroundings. Successively, evaporation results in crystallization of the matrix/analyte deposit on or close to the hydrophilic spot. This allows a much more reproducible interrogation of the sample since the laser spot covers a larger portion of the area coated with the matrix/analyte deposit. This eliminates the hunt for the “sweet spot”, while also increasing the sensitivity of the measurement due to the analyte concentration effect of the procedure (Schuerenberg, C. Luebbert, H. Eickhoff, M. Kalkum, H. Lehrach and E. Nordhoff: “Prestructured MALDI-MS sample supports”, Analytical Chemistry 72 (15), pp. 3436-3442 (2000)).
This procedure works reliably with water-soluble matrix compounds such as 2,5-DHP or 3-HPA, while it does not work well with water-insoluble matrix compounds such as HCCA (Schuerenberg, C. Luebbert, H. Eickhoff, M. Kalkum, H. Lehrach and E. Nordhoff: “Prestructured MALDI-MS sample supports”, Analytical Chemistry 72 (15), pp. 3436-3442 (2000); M. Schuerenberg: “AnchorChip™ Technology, Revision 2.3”, Bruker Product Information, (2005)). When HCCA is used, the final deposit is spread over an area much wider than the anchor spot. The reason for this behavior lies in the necessity to use an organic solvent mixable with water to dissolve the HCCA matrix. Usually, acetonitrile is used as organic solvent since it dissolves HCCA, and it is polar enough to mix well with the aqueous solution containing the analyte to be investigated.